Abstract

The human gut microbiome is a complex ecosystem composed mainly of uncultured bacteria. It plays an essential role in the catabolism of dietary fibers, the part of plant material in our diet that is not metabolized in the upper digestive tract, because the human genome does not encode adequate carbohydrate active enzymes (CAZymes). We describe a multi-step functionally based approach to guide the in-depth pyrosequencing of specific regions of the human gut metagenome encoding the CAZymes involved in dietary fiber breakdown. High-throughput functional screens were first applied to a library covering 5.4 × 10(9) bp of metagenomic DNA, allowing the isolation of 310 clones showing beta-glucanase, hemicellulase, galactanase, amylase, or pectinase activities. Based on the results of refined secondary screens, sequencing efforts were reduced to 0.84 Mb of nonredundant metagenomic DNA, corresponding to 26 clones that were particularly efficient for the degradation of raw plant polysaccharides. Seventy-three CAZymes from 35 different families were discovered. This corresponds to a fivefold target-gene enrichment compared to random sequencing of the human gut metagenome. Thirty-three of these CAZy encoding genes are highly homologous to prevalent genes found in the gut microbiome of at least 20 individuals for whose metagenomic data are available. Moreover, 18 multigenic clusters encoding complementary enzyme activities for plant cell wall degradation were also identified. Gene taxonomic assignment is consistent with horizontal gene transfer events in dominant gut species and provides new insights into the human gut functional trophic chain.

CAZy gene clusters for each clone sequence from 1 to 26. Below the clone number is the activity for which each clone has been screened. (Blue) CAZy-encoding genes; (yellow) SusD homolog–encoding genes; (green) transport system protein–encoding genes; (purple) other genes. 14/15 shows the CAZy gene clusters of assembled sequences from these clones. Clones 10 and 11 and clones 17 and 18 have the same CAZy gene clusters; these sequences are not assembled together. On top of each bar is the taxonomic assignation of the clone when assignable, other clones are nonassigned. (*) Synteny with Roseburia intestinalis L1-82 (1); Bacteroides uniformis ATCC 8492 (2); Bacteroides stercoris ATCC 43183 (3); Bacteroides eggerthii DSM 20697 (4).

Evidence of horizontal gene transfers (HGTs) in human gut metagenomic sequences. HGTs were identified when rupture was observed in gene synteny between the genes present in the metagenomic DNA fragments and their best BLASTP hits issued from sequenced genomes. For each clone, the first line represents the clone metagenomic sequence, and the second line represents the genome part in synteny with it. Each arrow represents a gene. (Red arrows) Genes encoding putative transposases or integrases; (black arrows) CAZy-encoding genes; (stars within black arrows) genes encoding the CAZymes involved in the activity detected in the primary screens, as proven by transposon insertion in the fosmid inserts.